Woodchip-sulfur based heterotrophic and autotrophic denitrification (WSHAD) process for nitrate contaminated water remediation

Water Research 89 (2016) 171e179

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Woodchip-sulfur based heterotrophic and autotrophic denitrification (WSHAD) process for nitrate contaminated water remediation Rui Li a, b, Chuanping Feng a, *, Weiwu Hu c, Beidou Xi b, d, **, Nan Chen a, Baowei Zhao d, Ying Liu a, Chunbo Hao a, Jiaoyang Pu a a

School of Water Resources and Environment, China University of Geosciences (Beijing), No. 29 Xueyuan Road, Haidian District, Beijing 100083, China State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China The Journal Center, China University of Geosciences, Beijing 100083, China d School of Environmental and Municipal Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 August 2015 Received in revised form 12 November 2015 Accepted 17 November 2015 Available online xxx

Nitrate contaminated water can be effectively treated by simultaneous heterotrophic and autotrophic denitrification (HAD). In the present study, woodchips and elemental sulfur were used as co-electron donors for HAD. It was found that ammonium salts could enhance the denitrifying activity of the Thiobacillus bacteria, which utilize the ammonium that is produced by the dissimilatory nitrate reduction to ammonium (DNRA) in the woodchip-sulfur based heterotrophic and autotrophic denitrification (WSHAD) process. The denitrification performance of the WSHAD process (reaction constants range from 0.05485 h
1 to 0.06637 h
1) is better than that of sulfur-based autotrophic denitrification (reaction constants range from 0.01029 h
1 to 0.01379 h
1), and the optimized ratio of woodchips to sulfur is 1:1 (w/w). No sulfate accumulation is observed in the WSHAD process and the alkalinity generated in the heterotrophic denitrification can compensate for alkalinity consumption by the sulfur-based autotrophic denitrification. The symbiotic relationship between the autotrophic and the heterotrophic denitrification processes play a vital role in the mixotrophic environment. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Denitrification Autotrophic Heterotrophic Mixotrophic Sulfur Woodchips

1. Introduction Nitrate, that occurs naturally as a part of the nitrogen cycle, is a ubiquitous contaminant of natural water resources. Several point and non-point sources such as synthetic and natural fertilization, bacterial production, atmospheric deposition, and leaking septic systems cause nitrate contamination. Nitrate has many detrimental effects: if ingested by infants, it causes methemoglobinemia, and when converted to nitrosoamines, it can cause carcinoma, malformation and mutation (Bordeleau et al., 2008). According to the World Health Organization (WHO), the maximum nitrate concentration in drinking water should be 50 mg L
1 (i.e., a nitratenitrogen value of 11.3 mg L
1) (WHO, 2008). In China, the maximum permissible concentration for nitrate in drinking water * Corresponding author. School of Water Resources and Environment, China University of Geosciences (Beijing), Beijing 100083, China. ** Corresponding author. State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China. E-mail addresses: [email protected] (C. Feng), [email protected] (B. Xi). http://dx.doi.org/10.1016/j.watres.2015.11.044 0043-1354/© 2015 Elsevier Ltd. All rights reserved.

is 10 mg L
1 of nitrate-nitrogen (i.e., a nitrate value of 44.2 mg L
1) (Zhang et al., 2013). To meet these stringent standards, it is important to develop efficient treatment processes for nitratecontaminated drinking water. Nitrate contaminated drinking water is commonly treated by methods such as reverse osmosis, ion exchange, distillation, and electrodialysis. These methods have a high operational cost, low selectivity, and can result in secondary brine wastes (Sahinkaya and Dursun, 2012). Biological denitrification has been considered as a suitable alternative process. In this process, heterotrophic denitrifiers utilize (added) organic substrates that serve as electron donors, and convert nitrate into nitrogen under anoxic conditions, achieving quite a high denitrifying rate and treatment capacity. However, nitrite can accumulate if the organic substrate does not fulfill the stoichiometric requirement. Conversely, if the organic substance added is too high, the residual amount can cause secondary pollution (Wen et al., 2003). Autotrophic denitrification is an alternative to heterotrophic denitrification for treating nitrate contaminated water. Since no organic substrates need to be added, bacterial contamination and operational costs are lower (Sahinkaya

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and Dursun, 2012). Autotrophic denitrification uses sulfur d which is non-toxic, water insoluble and stable under normal conditions d as an electron donor (Soares, 2002). However, the main disadvantage of the sulfur-based autotrophic denitrification (SAD, Eq. (3)) is the production of sulfate and other acids. The alkali consumed in the process is neutralized by adding limestone or other alkalinity source (Zhou et al., 2011) but this increases the hardness of the treated water. Therefore, the heterotrophic and autotrophic denitrification (HAD, mixotrophic) process has been developed to limit sulfate formation (Sahinkaya et al., 2011) and alkali consumption (Liu et al., 2009). This process synergizes products and reactants of heterotrophic and autotrophic denitrification: the former generates alkalinity which the latter consumes; the result is an alkalieneutral reaction (Oh et al., 2001). In the past decade, many studies investigated the HAD process (Liu et al., 2009; Oh et al., 2001; Qambrani et al., 2013; Sahinkaya and Dursun, 2012; Sahinkaya et al., 2011). They used liquid carbon substrates such as methanol (most common), sodium acetate, glucose, or molasses, and achieved lower sulfate levels and higher denitrification rates than SAD. The alternative to this, is to use solid carbon sources. Sawdust or woodchips are the most commonly used solid carbon substrates successfully applied in field-scale projects (Schmidt and Clark, 2012). The main available biological carbon in sawdust or woodchips is lignocellulosic materials. Some lignocellulolytic enzymes, e.g. cellulases, hemicellulases, and lignases etc., that can either release cellulose from the plant polymer lignin to increase sugar yields from biomass, or facilitate lignin transformation to biobased products (e.g. biofuel) (DeAngelis et al., 2010). Sawdust or woodchips based heterotrophic denitrification (HD) processes have long service lives (5e15 years), high nitrate removal rates (1e20 g N d
1 m
3media), and low maintenance costs (Robertson, 2010). It does have a drawback though: ammonium is released as an intermediate during the HD process (Yang et al., 2012) due to dissimilatory nitrate reduction to ammonium (DNRA) (Patterson et al., 2002). Zhang et al. (2012) also observed ammonium accumulation when sawdust was used as the solid carbon substrate for denitrification. Furthermore, ammonium was also considered to be an intermediate product in the denitrification process (Yang et al., 2012). Early in 1954, research showed that Thiobacillus bacteria grew better with added ammonium salts (Baalsrud and Baalsrud, 1954). Till date, however, no concrete evidence has confirmed the necessity of ammonium salts. While it seems probable that the ammonium produced during HD can be utilized by Thiobacillus in HAD, this has yet to be tested. Furthermore, it has been found that the HAD bio-process have certain potential in the field of wastewater treatment, while both nitrate and toxic nitrite in the wastewater can be effectively removed (Li et al., 2015). In the present study, woodchips and sulfur were selected as corporate electron donors for the HAD process. Batch experiments were conducted to investigate the synergistic effect between HD and SAD. The denitrification performance, sulfate production, pH variation, and ammonium utilization were analyzed, and DGGE and 16S rRNA-based microbial analyses were conducted to give insight into the microbiota. 2. Materials and methods 2.1. Preparation of seed sludge Seed sludge was obtained from a pre-anoxic zone in the Qinghe Wastewater Treatment Plant, Beijing, China. The sludge was stored in a sealed 2 L beaker at room temperature (20 ± 2  C) for 48 h. The suspended solids (SS; 5028 mg L
1), volatile suspended solids (VSS; 4137 mg L
1), oxidation reduction potential (ORP;
279.1 mv),

dissolved oxygen (DO; 0.09 mg L
1), and pH (7.09) in the sludge were measured. The ratio of VSS/SS was approximately 0.82. One L of the sludge mixture was transferred into a 1 L beaker. After allowing it to settle for 30 min, the supernatant was replaced with a liquid nutrient medium suitable for cultivating Thiobacillus bacteria. The nutrient medium (pH ~ 7) included the following components at the indicated concentrations: Na2S2O3$5H2O (20 mM), NaHCO3 (30 mM), KNO3 (20 mM), KH2PO4 (14.7 mM), NH4Cl (18.7 mM), MgSO4$7H2O (3.25 mM), FeSO4$7H2O (0.08 mM) and CaCl2$2H2O (0.05 mM) (Beller, 2005). The medium was replaced every day. Cultures were cultivated at 30  C for 20 d. Meanwhile, heterotrophic denitrifiers were also cultivated by the same process but with a different liquid nutrient medium that contained: CH3COONa (33.28 mM), NH4Cl (5.72 mM), KNO3 (9.89 mM), KH2PO4 (1.10 mM), MgSO4$7H2O (0.41 mM), FeSO4$7H2O (0.02 mM), pH 7.0e7.3 (Yao et al., 2013). 2.2. Batch experiments To investigate the WSHAD process, eight batch experiments were designed. Each batch experiment was carried out in duplicate in 500 mL serum bottle bioreactors. The dosages of sulfur, NH4Cl, and limestone in the bioreactors are shown in Table 1. The dosage ratios (w/w) of sulfur to NH4Cl in the bioreactors SLNmin, SLNmid, and SLNmax were 10:0.61, 10:1.22, and 10:1.83, with NH4Cl dosage being minimum, middle, and maximum respectively. The dosage ratio of woodchips to sulfur (w/w) in the bioreactors W, WSin, WSst, and WSex were 1:0, 1:0.5, 1:1, and 1:1.5 with woodchip dosage being insufficient, stoichiometric, and excess respectively. The sulfur particles (particle size 4e6 mm) were purchased from the Hengtianyi chemical factory co., Ltd, Beijing, China. The S content in the sulfur particle was 99.334 ± 0.380% as measured by an elemental analyzer (Flash 2000, Thermo Fisher, Italy). For its wide distribution and low cost in china, pine woods are more frequently used as raw material for manufacturing than other hard woods. In consideration of resources recovery and the potential for serving as carbon source in the sulfate reducing bio-process (Roman et al., 2008), the pine woodchips were purchased from Hengshui city, Hebei, China, and were sieved to get particle size of 2.5e10 mm. The C, H, N, and S content in the woodchips were 47.090 ± 0.465%, 4.803 ± 0.223%, 0.387 ± 0.021%, and 0.345 ± 0.033% as measured by the elemental analyzer mentioned above. The NH4Cl and limestone were of analytical grade. 2.3. Inoculation Before inoculation, supernatants (liquid nutrient medium) in the two seed sludge cultivation beakers were discarded; the remaining cultures were washed thrice with sterile physiological saline (to remove most of the residual nitrate and sulfate), and diluted to 1 L with deionized water. The beakers were stirred homogeneously for 1 min, after which 100 mL of the autotrophically acclimated cultures and 100 mL deionized water were immediately transferred into SL, SLNmin, SLNmid, and SLNmax; 100 mL of heterotrophic denitrifier cultures and 100 mL deionized water were transferred into the W; 100 mL of the autotrophically acclimated cultures and 100 mL of the heterotrophic denitrifier cultures were transferred into WSin, WSst, and
1 WSex, and 300 mL of solution containing NO
3 (60 mg N L ) and 2

1 PO4 (12 mg P L ), was transferred into each bioreactor. Ultimately, the initial nitrate concentration in each bioreactor was ~36 mg N L
1. All the bioreactors were purged with N2 gas for 15 min to create anoxic conditions, and incubated at 30  C (Qambrani et al., 2013) in a thermostatic bath oscillator (DDHZ-300, Taicang Experimental Equipment Factory, China).

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Table 1 The dosage of sulfur, NH4Cl, limestone, and woodchips in the batch experiments. Bioreactor

Sulfur (g)

NH4Cl (g)

Limestone (g)

Woodchips (g)

Experimental purposes

SL SLNmin SLNmid SLNmax W WSin WSst WSex

10 10 10 10 0 5 10 15

0 0.61 1.22 1.83 0 0 0 0

15 15 15 15 0 0 0 0

0 0 0 0 10 10 10 10

No ammonium control Minimum ammonium Middle ammonium Maximum ammonium Woodchips control Insufficient sulfur Stoichiometric sulfur Excessive sulfur

2.4. Analysis A volume of 5 mL supernatant was periodically withdrawn from each bioreactor. The pH of the sample was measured immediately using a pH meter (Seven Multi S40, Mettler Toledo, Switzerland). ATP (adenosine triphosphate) in the cultures was measured by an ATP fluorescence detector (AF-100, TOADKK, Japan). Each sample was filtered through 0.45 mm membrane filter, and then diluted to one-tenth the concentration with deionized water.
Sulfate, NO
3 , and NO2 were measured by an ion chromatograph (ICS900 Dionex IonPac, Thermo Fisher Scientific, US) (Pu et al., 2014). Two mL of diluted sample were treated with 0.5 mL of H2O2 (3%, v/v) to oxidize sulfite and thiosulfate in the samples to sulfate, and then the total sulfate was measured. The NHþ 4 was measured using a spectrophotometer (DR6000, HACH, US) according to the Water and Wastewater Monitoring Analysis Method (SEPA, 2002). Paired-Samples T-Test in IBM SPSS Statistics 19 was undertaken for analyzing significant differences between batch experiments. 2.5. DGGE and 16S rRNA based microbial analysis DGGE analysis was performed by Bio-Rad C-1000 system (BioRad, USA). The PCR products were loaded in the gel, which consisted of 1 mm-thick 9% (w/v) polyacrylamide and had a denaturant gradient of 40%e60% (100% denaturant was 7 M urea and 40% formamide). The electrophoresis was run in 1  TAE (20 mM Tris, 10 mM acetate, 0.5 mM EDTA, pH 8.0) at 60  C for 12 h at 100 V. The gel was then stained with staining dye solution and placed on a UV illuminator (Bio-Rad, USA). The excised gel from each target band was placed in a sterile 1.5 mL tube containing 30 mL of sterile ultrapure water and crushed. 1 mL of eluted DNA was amplified with the primer 968F/1401R. The DNA was sequenced and sequencing was carried out by Shanghai Sangon Co., Ltd., China. The nucleotide sequences reported in this paper have been submitted to GenBank with accession numbers: KM379101eKM379114. For 16S rRNA-based microbial analysis, genomic DNA was extracted and amplified with 968F GC and 1401R primers, using the PCR system (initial denaturation, 95  C for 5 min; subsequent denaturation, 95  C for 0.5 min; annealing, 54  C for 0.5 min; extension, 72  C for 45 s and final extension, 72  C for 10 min). Another round of PCR was performed with amplified 16S rRNA genes. 3. Results and discussion 3.1. Nitrate removal The bioreactors SL, SLNmin, SLNmid, and SLNmax were designed to study the influence of artificially added ammonium on the SAD. According to Eq. (3), the stoichiometric ratio of sulfur to NH4Cl is 10:1.22 (w/w). As shown in Fig. 1a, nitrate concentrations in SL,

SLNmin, SLNmid, and SLNmax continuously declined until 192 h. Nitrate concentration in SL, SLNmin, SLNmid, and SLNmax decreased from 36.63 ± 1.83, 36.71 ± 1.65, 36.71 ± 1.65, and 36.51 ± 1.39 mg N L
1 at 0 h to 6.82 ± 0.34, 7.03 ± 0.32, 6.39 ± 0.26, and 7.20 ± 0.27 mg N L
1 at 192 h, respectively. Although initially at the same level, after 0.5 h, the nitrate concentrations in SLNmin, and SLNmid were lower than that in SL (p < 0.05, as shown in supporting information), the nitrate concentration difference between SL and SLNmax were not significant (p > 0.05), and the trends in the SLNmin and SLNmax were similar (p > 0.05). SLNmid had a continuously lower nitrate concentration than SLNmin and SLNmax (p < 0.05), indicating that insufficient or excess dosage of ammonium (SLNmin or SLNmax) had a negative impact on nitrate removal. It was suggested that Thiobacillus denitrificans could grow normally with 0.0083%e0.083% NH4Cl in a previous nitrateethiosulfate medium (Baalsrud and Baalsrud, 1954). Improper NH4Cl dosage in the present sulfur-ammonium-limestone bio-system might give rise to nutrient element imbalance (e.g. N:S ratio), which was crucially important for the microbial activity. As shown in Table 2, the nitrate concentration trends followed first-order kinetics. The reaction constant k1 in the bioreactors with added ammonium was higher than that in the bioreactor without ammonium. This confirms the findings of literature (Baalsrud and Baalsrud, 1954), that ammonium salts benefit the metabolic activity of the Thiobacillus bacteria in the SAD process. The activity and viable count of microorganisms were characterized by ATP activities. ATP activity increments in SLNmin, SLNmid, and SLNmax were higher than that in SL until 192 h (data not shown), indicating that the ammonium salts were beneficial to denitrifying activity of microorganisms in the SAD. Furthermore, ATP activity in SLNmid increased more than that in SLNmin and SLNmax, suggesting that the stoichiometric dosage ratio (10:1.22, w/w) of sulfur to NH4Cl in SAD might enhance the bioactivity. The bioreactors W, WSin, WSst, and WSex were designed to study the WSHAD process. Nitrate concentrations sharply decreased in the first 48 h (Fig. 1a). Nitrate concentrations in W, WSin, WSst, and WSex decreased rapidly from 36.48 ± 1.64, 36.65 ± 1.69, 36.72 ± 1.32, and 36.67 ± 1.65 mg N L
1 at 0 h to below detection limits (<0.02 mg N L
1), resulting in a much higher nitrate removal rate than the SAD process (p < 0.05). This is in line with previous findings (Qambrani et al., 2013). As shown in Table 2, the nitrate concentration variation trends in the W, WSin, WSst, and WSex also obeyed first-order kinetics the reaction constant k1 being higher than the HD process, indicate higher nitrate removal rates of the WSHAD process. However, the statistical differences of nitrate concentrations between W, WSin, WSst, and WSex were not significant (p > 0.05). The fraction of nitrate denitrified by autotrophic or heterotrophic denitrification could be determined indirectly from the production of sulfate (Oh et al., 2001). Sulfate produced in WSin, WSst, and WSex at 48 h were 20.93 ± 0.75, 35.94 ± 1.29, and 37.24 ± 1.19 mg L
1 (Fig. 2a). According to Eq. (3), stoichiometric value of nitrate reduced autotrophically in WSin, WSst, and WSex

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þ Fig. 1. Denitrification performances showing (a) NO
3 , (b) NO2 and (c) NH4 profiles with time in the batch experiments in the bioreactors.

Table 2 Reaction constants for first-order kinetics in the batch experiments. First-order kinetics equation: c ¼ c0 $ e
k1
1

k1 (h SL SLNmin SLNmid SLNmax W WSin WSst WSex

)

0.01029 0.01108 0.01379 0.01106 0.05493 0.05485 0.06637 0.05885

R

2

0.9907 0.9815 0.9271 0.9702 0.9904 0.9923 0.9948 0.9880

$ ðt
t0 Þ

SE 3.34  10
4 4.90  10
4 0.00122 6.09  10
4 0.00336 0.00293 0.00304 0.00403

should be 2.77, 4.76, and 4.94 mg N L
1; i.e. 7.57%, 12.98%, and 13.46% of initial nitrate in WSin, WSst, and WSex were reduced by the autotrophic denitrification respectively. However, the stoichiometric fraction of autotrophic denitrification might be lower than that in practice, due to the possibility of sulfate reducing process before 48 h.

3.2. Nitrite accumulation As shown in fig. 1b, nitrite concentrations in SL, SLNmin, SLNmid, and SLNmax continuously increased until 192 h. Nitrite concentration in SL, SLNmin, SLNmid, and SLNmax increased to 3.06 ± 0.15,

R. Li et al. / Water Research 89 (2016) 171e179

175

Fig. 2. Sulfate and pH variations in the batch experiments.

1.85 ± 0.04, 1.75 ± 0.09, and 2.03 ± 0.10 mg N L
1 at 192 h with SL having the highest concentration throughout. Nitrite therefore accumulated in the absence of ammonium (p < 0.05), further confirming that ammonium salts could enhance the denitrifying activity of Thiobacillus bacteria in the SAD. The nitrite concentration in SLNmid was lower than that in SLNmin and SLNmax (p < 0.05), and the stoichiometric dosage ratio (10:1.22, w/w) of sulfur to NH4Cl in SLNmid could be the reason for lower nitrite accumulation. Transient nitrite accumulation (3.5 mg N L
1 e 4 mg N L
1) was observed when nitrate breakthrough occurred in a SAD membrane bioreactor during the 100 days operation, which could be overcome by sulfur compensation (Sahinkaya et al., 2015). Nitrite formation was arisen in a SAD column bio-system when the influent nitrate concentration increased (Sahinkaya et al., 2014). These results indicated once again that nutrient element balance of N:S was one of the key factors for the SAD process. Four key enzymes are

responsible for nitrogen oxide reduction in the denitrification process, namely, nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase. These enzymes are usually induced sequentially under anaerobic conditions (Shao et al., 2011). Incomplete denitrification could be the reason for the nitrite accumulation. The nitrite concentration in W, WSin, WSst, and WSex increased to 18.75 ± 0.52, 10.97 ± 0.25, 8.56 ± 0.20, and 9.68 ± 0.18 mg N L
1 until 24 h, and then decreased to below detection limits (<0.009 mg N L
1) at 48 h. Nitrite accumulation in W was the most severe at 24 h (Fig. 1b), and in WSst it was slightly lower than that in WSin and WSex (p < 0.05). Nitrite is an intermediate in the denitrification process, reduced from nitrate at high nitrate concentrations. Nitrite reductase produced by nitrite-reducing microorganisms reduces nitrite when the nitrate concentration is below the inhibitory level (500 mg N L
1) in a thiosulfate based

176

R. Li et al. / Water Research 89 (2016) 171e179

autotrophic denitrification process (Chung et al., 2014). In this study, perhaps the nitrate inhibitory levels for nitrite reductases in W, WSin, WSst, and WSex were 8.79 mg N L
1, 12.09 mg N L
1, 6.57 mg N L
1, and 7.71 mg N L
1 respectively at 24 h, and thus nitrite accumulation peaked at the same time. The inhibitory level was observed approximately 3 mg N L
1 - 16 mg N L
1 in a HAD biosystem which utilized liquid carbon source (Qambrani et al., 2013). A denitrification system with higher nitrate inhibitory level is more appropriate for severely nitrate contaminated water treatment; however, biomass yields might be much more in this situation while back wash technology is needed. The HAD bioreactor with low nitrate inhibitory level can maintain favorable hydraulic conductivity, and might be more suitable for application in in-situ groundwater treatment (e.g. Permeable Reactive Barrier technology). In 192 h, nitrite accumulation in the SAD processes continued to increase, whereas it was ultimately eliminated in the HD and WSHAD processes. This difference may be ascribed to the different nitrate removal efficiencies. As shown in Table 2, the nitrate removal efficiencies in the WSHAD bioreactors were higher than those in SL, SLNmin, SLNmid, and SLNmax. Therefore, the HD and WSHAD processes had higher nitrate removal rates and less nitrite accumulation in the denitrification processes. 3.3. Ammonium accumulation The initial ammonium concentration was undetectable (<0.019 mg N L
1) in SL, 949.67 ± 18.04 mg N L
1 in SLNmin, 1901.67 ± 36.13 mg N L
1 in SLNmid, and 2845.89 ± 85.38 mg N L
1 in SLNmax. The ammonium concentration in SL increased to 0.09 ± 0.003 mg N L
1 at 144 h. Afterwards, the ammonium concentration decreased to 0.05 ± 0.003 mg L
1 at 192 h. In this period, the autotrophic denitrifiers might have consumed the 36.36% of the ammonium produced by the DNRA process. As shown in Fig. 1c, the ammonium concentrations in SLNmin, SLNmid, and SLNmax at 0.5 h were 15.53 ± 0.53, 12.78 ± 0.37, and 10.26 ± 0.26 mg N L
1, leading to a 98.4%, 99.3%, and 99.6% reduction of ammonium respectively. The chemical reaction between the NH4Cl and limestone (CaCO3) could be the main reason for the significant ammonium reduction in the first 0.5 h (Eq. (1) and Eq. (2)). However, due to the NH4Cl dosing, residual ammonium concentrations in SLNmin, SLNmid, and SLNmax were higher than that in SL. The ammonium carbonate produced in the first 0.5 h might also be utilized by the Thiobacillus bacteria for metabolic activity (Aziz et al., 2004).

NH4 Cl þ H2 O # NH3 $H2 O þ HCl

(1)

CaCO3 þ 2HCl / CaCl2 þ H2 O þ CO2

(2)

Until 120 h, ammonium concentrations in SLNmin, SLNmid, and SLNmax increased slowly. Ammonium can be produced by the DNRA process in sulfur or sulfide based autotrophic denitrification (Brunet and GarciaGil, 1996), thus the increase in ammonium concentrations might be attributed to it. After 120 h, ammonium concentrations decreased to 14.66 ± 0.60, 11.92 ± 0.43, and 13.00 ± 0.65 mg N L
1 at 192 h, and this could be due to the utilization of ammonium salts by the denitrifiers. Approximately 21.2%, 28.7%, and 6.1% of ammonium decreased from 120 h to 192 h. Ammonium decreased more in SLNmid than in SLNmin and SLNmax, indicating that the stoichiometric dosage ratio (10:1.22, w/w) of sulfur to NH4Cl was effective in reducing ammonium accumulation. In W, the ammonium concentration increased slowly to 0.07 ± 0.002 mg N L
1 at 120 h, and then increased rapidly to 0.24 ± 0.007 mg N L
1 at 192 h. The DNRA in the heterotrophic

denitrification could also be the reason for the ammonium accumulation (Zhang et al., 2012). Ammonium could also be released by mineralization of the organic N resulting from microbial activities under anaerobic conditions, while ammonium releasing was not observed obviously in the woodchips based HD process (Greenan et al., 2006). The ammonium concentration in WSin increased to 0.27 ± 0.011 mg N L
1 at 96 h, while the concentration in WSst and WSex increased to 0.12 ± 0.005 and 0.19 ± 0.008 mg N L
1 at 120 h. In this period, ammonium accumulation levels in WSin, WSst, and WSex were higher than that in W, after which the ammonium concentration in WSin, WSst, and WSex decreased to 0.10 ± 0.002, 0.02 ± 0.001, and 0.13 ± 0.003 mg N L
1 at 192 h. Around 61.9%, 80.0%, and 31.7% of ammonium was utilized in WSin, WSst, and WSex, indicating that the ammonium produced by the DNRA process could be utilized by the autotrophic denitrifiers in the HAD process. Nevertheless, statistical differences of ammonium accumulations between most of the batch experiments were not significant (supporting information). 3.4. Sulfate accumulation The sulfate concentration in SL, SLNmin, SLNmid, and SLNmax increased continuously to 262.13 ± 11.01, 311.05 ± 15.86, 281.84 ± 12.96, and 295.96 ± 12.43 mg L
1 until 192 h (Fig. 2a). According to the SAD process (Eq. (3)), sulfate production in SL, SLNmin, SLNmid, and SLNmax should be 1.171 mmol, 1.166 mmol, 1.190 mmol, and 1.151 mmol respectively, at 192 h. According to sulfate concentrations measured at 192 h, 0.13 g, 0.16 g, 0.14 g and 0.15 g of sulfate are produced in 500 mL bioreactor of SL, SLNmin, SLNmid, and SLNmax respectively. For relative molecular weight of sulfate is 96 g mol
1, 1.365 mmol, 1.620 mmol, 1.468 mmol, and 1.541 mmol of sulfate could be produced in each bioreactor. A series of intermediate products such as sulfite and thiosulfate can be formed during the SAD process (Pu et al., 2014). On the other hand, elemental sulfur could be transformed to sulfide due to the sulfur disproportionation process, when the electron donor was limited (Sahinkaya et al., 2015). However, a sulfate level close to the total sulfate was detected at 192 h in this study (data not shown), indicating that the intermediate products (sulfite, thiosulfate, and sulfide) might be oxidized to sulfate during the SAD process. During the SAD process, the sulfate production may not completely obey the stoichiometric relationship (Eq. (3)) due to these intermediate reaction processes. The sulfate production in SLNmin, SLNmid, and SLNmax was higher than that in SL (p < 0.05), because the nitrate removal efficiency was a little higher (Fig. 1a). In contrast, nitrate removal efficiency in SLNmid was slightly higher than that in SLNmin and SLNmax, while the sulfate production in SLNmid was relatively lower (p < 0.05). That might be due to the differing microbial environment affecting the solubility of CaCO3. The sulfate concentration in W increased slowly to 6.32 ± 0.12 mg L
1 at 24 h, and then decreased below 0.09 mg L
1 (detection limit) at 72 h, with no further changes. Since elemental S was measured in the woodchips used in this study, sulfate might have been produced from the woodchips before 72 h. The sulfate concentration in WSin, WSst, and WSex increased to maximum values of 31.34 ± 0.72 mg L
1 at 36 h, 35.94 ± 1.29 mg L
1 at 48 h, and 38.37 ± 1.92 mg L
1 at 24 h. In WSin and WSst, it then decreased below 0.09 mg L
1 (detection limit) at 96 h and 120 h respectively; and in WSex, it decreased to 2.88 ± 0.12 mg L
1 at 144 h. Further, no changes were observed. Sulfate accumulation thus decreased in the WSHAD processes. Sulfate can be reduced by sulfate-reducing bacterial communities in anaerobic conditions. In this dissimilatory process, organic matter serves as an electron donor, terminally producing hydrogen

R. Li et al. / Water Research 89 (2016) 171e179

sulphide (H2S) (Jørgensen, 1982). It has been reported that some facultative autotrophic denitrifiers use sulfur compounds as energy sources and adapt to different environments (i.e. autotrophic, heterotrophic, or mixotrophic conditions) (Matin, 1978). The sulfate-reducing bacteria and the sulfur based autotrophic denitrifiers were therefore probably symbiotic in the mixtrophic environment in WSin, WSst, and WSex, and the sulfate produced by the autotrophic denitrifiers was ultimately utilized by the sulfatereducing bacteria to produce H2S. This hypothesis will be confirmed by the DGGE and 16S rRNA-based microbial analysis further in this study. No sulfate accumulated in the WSin and WSst from 120 h to 192 h, unlike WSex, where it continued to accumulate (Fig. 2a). Excessive dosage of sulfur in WSex would result in much more sulfate accumulation in the WSHAD process (p < 0.05). The H2S generated during sulfate reduction process is an air pollutant, and its invasion of the atmosphere with a noxious odor makes the atmosphere unpleasant to humans (Liu et al., 2015). Accordingly, the produced H2S needs to be further treated (e.g. oxidized by the aerobic treatment process) or recycled (e.g. utilized for precipita
nez-Rodríguez tion of heavy metals such as Fe, Cu, Zn and Al) (Jime et al., 2009). 3.5. pH variations The pH in SL, SLNmin, SLNmid, and SLNmax decreased from 9.68 ± 0.03, 9.65 ± 0.01, 9.66 ± 0.02, and 9.63 ± 0.02 at 0 h to 7.61 ± 0.01, 7.56 ± 0.02, 7.54 ± 0.02, and 7.50 ± 0.01 at 72 h (Fig. 2b). Sufficient limestone addition could neutralize the alkali consumption in the SAD process. Moreover, the pH in SLNmin, SLNmid, and SLNmax at 0.5 h was lower than that in SL (p < 0.05), because NH4Cl added to the former could react with CaCO3, inhibiting the hydrolytic action of CaCO3 in water. The higher the dosage of NH4Cl in SLNmin, SLNmid, and SLNmax, the lower the measured pH (p < 0.05). The pH in W increased until 48 h, with no further significant change, indicating again that the heterotrophic denitrification is an alkalinity generating process. However, the pH in WSin, WSst, and WSex decreased until 72 h, with no further significant change. This is because the alkalinity consumption by sulfur-based autotrophic denitrification could neutralize the alkalinity generated in the heterotrophic denitrification. Therefore, the WSHAD process needs no alkali addition. 3.6. Bacterial community analysis As shown in Table 3, Thiobacillus denitrificans, Thiobacillus aquaesulis, Thiobacillus thioparus, Hydrogenophaga sp., and

177

Comamonas sp. were found in the SL and SLNmid. Thiobacillus denitrificans is an obligate chemolithoautotrophic and facultative anaerobic bacterium, well known for its ability to use sulfur as an electron donor for denitrification (Beller et al., 2006). Thiobacillus aquaesulis is a facultative chemolithotrophic bacterium that can grow on thiosulphate agar and in liquid batch culture (Wood and Kelly, 1988), and it was also present in WSst. Thiobacillus thioparus is also an obligate chemolithoautotrophic bacterium that can use sulfur as an electron donor for denitrification (Boden et al., 2012). Hydrogenophaga is a genus of hydrogen-oxidizing bacteria, that are chemo-organotrophic or chemolithoautotrophic (Kampfer et al., 2005), and some members of this genus were previously described as Pseudomonas species (Willems et al., 1989). Genus Pseudomonas is well known for its denitrification abilities. The Hydrogenophaga sp. present in SL and SLNmid and W and WSst might have originated from the seed sludge that was obtained from the primary anaerobic digester as mentioned above. The genus Comamonas is a family of genera of the family Comamonadaceae, of which some members can use the bacterial polyester poly (3hydroxybutyrate-co-3-hydroxyvalerate) as solid substrate for denitrification (Khan et al., 2002). The Acidobacteriaceae bacterium present in W and WSst is a member of Acidobacteria, and is symbiotic with some sulfatereducing bacteria belonging to the Firmicutes (Barberan et al., 2012). It was further shown that the sulfate-reducing bacteria in the WSHAD could ultimately utilize the sulfate produced by the autotrophic denitrifiers. The presence of the obligate chemolithoautotrophic bacterium Thiobacillus denitrificans in the heterotrophic (W) and mixotrophic environment (WSst), indicates that organic matter in the heterotrophic or mixotrophic environment could not inhibit growth of chemolithoautotrophic bacteria. Therefore, the Thiobacillus thioparus was also detected in the WSst. As shown in Fig. 3, the banding pattern of lanes A and B (SL and SLNmid) were similar and lane C represents the banding pattern of a heterotrophic bacterial community in W. Lane D (WSst) is a combination of the band patterns of lanes A, B, and C, indicating that the autotrophic bacteria (in SL and SLNmid) and the heterotrophic bacteria (in W) coexisted in the mixotrophic environment (WSst). 3.7. Stoichiometric coefficients and mechanism description The stoichiometric coefficients of the WSHAD process are given in Table 4, assuming that (i) the organic carbon in woodchips was completely hydrolyzed to C6H12O6, (ii) 90% C6H12O6 could participate in the denitrification process (Eq. (4)) while 10% C6H12O6 was utilized by the DNRA process (Eq. (5)), based on the results obtained by Schmidt and Clark (2012), and (iii) NHþ 4 produced in Eq.

Table 3 Bacterial species based on the DGGE profile. Sample location

Most closely related sequence

% Similarity

Phylogenetic group

SL and SLNmid

Thiobacillus denitrificans Thiobacillus aquaesulis Thiobacillus thioparus Hydrogenophaga sp. Comamonas sp. Thiobacillus denitrificans Acidobacteriaceae bacterium Hydrogenophaga sp. Comamonas sp. Thiobacillus denitrificans Acidobacteriaceae bacterium Thiobacillus aquaesulis Thiobacillus thioparus Hydrogenophaga sp. Comamonas sp.

98 99 99 97 97 98 95 97 97 98 95 99 99 97 97

Betaproteobacteria Betaproteobacteria Betaproteobacteria Betaproteobacteria Betaproteobacteria Betaproteobacteria Acidobacteria Betaproteobacteria Betaproteobacteria Betaproteobacteria Acidobacteria Betaproteobacteria Betaproteobacteria Betaproteobacteria Betaproteobacteria

W

WSst

178

R. Li et al. / Water Research 89 (2016) 171e179

hydrolyzing from woodchips would be theoretically involved; meanwhile, 0.08 mol organisms (C5H7O2N), 1.1 mol SO2
4 , and 0.92 mol NHþ could be produced. 4 þ 1:1S þ NO
3 þ 0:76H2 O þ 0:4CO2 þ 0:08NH4 /0:08C5 H7 O2 N þ þ 1:1SO2
4 þ 0:5N2 þ 1:28H

(3) þ 24NO
3 þ 5C6 H12 O6 þ 24H /12N2 þ 30CO2 þ 42H2 O

(4)

þ þ 3NO
3 þ C6 H12 O6 þ 6H /3NH4 þ 6CO2 þ 3H2 O

(5)

þ 10NO
3 þ 1:1S þ 2C6 H12 O6 þ 8:72H / 0:08C5 H7 O2 N þ þ 1:1SO2
4 þ 0:92NH4 þ 4:5N2 þ 11:6CO2 þ 14:24 H2 O

(6)

4. Conclusions

Fig. 3. DGGE profile of bacterial community (Lanes A, B, C, and D show profiles from SL, SLNmid, W, and WSst respectively).

Table 4 Stoichiometric coefficients in the WSHAD process calculated based on the assumptions that (i) the organic carbon in woodchips was completely hydrolyzed to C6H12O6, (ii) 90% C6H12O6 could participate in the denitrification process (Eq. (4)) while 10% C6H12O6 was utilized by the DNRA process (Eq. (5)) and (iii) NHþ 4 produced in Eq. (5) could be utilized by the Eq. (3). All units are in moles. Eq. 3

NO
3 1 3.75

S 1.1 4.13

NHþ 4 0.08 0.30

Eq. 4

NO
3 24 4.32

C6H12O6 5 0.9

Hþ 24 4.32

/

NO
3 3 0.3

C6H12O6 1 0.1

Hþ 6 0.6

/

Eq. 5

CO2 0.4 1.50

/

Hþ 1.28 4.80

Ammonium salts can enhance the denitrifying activity of Thiobacillus bacteria in sulfur-based autotrophic denitrification. The denitrification performance of the woodchip-sulfur based mixotrophic process is better than that of sulfur-based autotrophic denitrification, and the optimum ratio of woodchips to sulfur is 1:1 (w/w). The sulfate-reducing bacteria utilized the sulfate produced by the autotrophic denitrifiers, and the alkalinity generated in the heterotrophic denitrification could effectively compensate for alkalinity consumption by the autotrophic denitrification. The DGGE and 16S rRNA-based microbial analysis revealed that the autotrophic and heterotrophic bacteria coexisted in the mixotrophic environment. Acknowledgments This study was supported by the National Natural Science Foundation of China (NSFC) (No.51578519), the Foundation for the Advisor of Beijing Excellent Doctoral Dissertation (No. 20121141501, No. 20131141502) and the Fundamental Research Funds for the Central Universities (No. 2652015122). Appendix A. Supplementary data

CO2 30 5.4 NHþ 4 3 0.3

CO2 6 0.6

(5) could be utilized by the Eq. (3). The Hþ generated in Eq. (3) was 4.8 mol, which could fulfill the Hþ requirement in Eq. (4) (4.32 mol) and Eq. (5) (0.6 mol). Based on these stoichiometric calculations, the stoichiometric ratio of woodchips to sulfur should be 1.30:1.13 (w/w). The 1:1 (w/w) dosage ratio of woodchips to sulfur in WSst was rather close to the stoichiometric ratio. The nitrate removal rate of WSst was thus higher than that of WSin and WSex (Table 2). The ATP concentration in WSst was higher than that in WSin and WSex at 192 h (data not shown), suggesting that the stoichiometric dosage ratio (1:1, w/w) of sulfur to woodchips in WSHAD was necessary. The overall stoichiometric equation of the WSHAD process (Eq. (6)) was proposed according to Eq. (3), Eq. (4), and Eq. (5). Briefly, if 10 mol nitrate participated in the bio-process, 8.72 mol Hþ, 1.1 mol bio-available S, and 2 mol C6H12O6

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